Quantitative rate and equilibrium constants for the hydrolysis of the lactone (ring E) in camptothecin (1) and analogues (2-5, all substituted in the A ring of 1) at 25 degrees C in aqueous solution were determined by high-performance liquid chromatography with UV-visible detection and by UV-visible spectrophotometry. It was shown that the lactone was converted to the carboxylate in a pH-dependent equilibrium. No major differences were observed in rate and equilibrium constants for 1-5, a result suggesting that the mechanism of lactone hydrolysis is independent of a variety of substituents on the A ring. The conversion of the lactone to its carboxylate form occurred under neutral and basic conditions and appeared to be largely dependent on hydroxide ion. The conversion of the carboxylate to the lactone was observed under neutral and acidic conditions and was pH independent at pH values greater than 5 and dependent on hydronium ion at pH values less than 5. Significant incorporation of oxygen-18 into the lactone ring of 3, a water-soluble analogue of 1, was observed for the ring opening and subsequent ring closing of 3 in H2(18)O. This finding strongly suggests that the mechanism of lactone ring hydrolysis involved acyl cleavage rather than alkyl cleavage. Kinetic solvent isotope effects (kH2O/kD2O, where kH2O and kD2O are the rate constants in H2O and D2O, respectively), obtained over the pH-rate profile for 1, were used to speculate about the nature of the transition states involved in the opening and closing reactions of the lactone ring.